Universal flu vaccine test in pigs

Dr. Paul Heinen

Facultad de Ciencias Exactas y Naturales

Universidad Nacional de Asunción

paulheinen@yahoo.com.ar

Vaccination of pigs with a DNA construct expressing

an influenza M2-nucleoprotein fusion protein

exacerbates disease after challenge with influenza A virus

Heinen, P.P., Rijsewijk, F.A., De Boer-Luijtze, E.A., Bianchi, A.T.J.

J Gen Virol 2002, 83, 1851-1859

Graduate School of Animal Health, Utrecht University,

detached at the Institute for Animal Science and Health (ID-

Lelystad), Department of Mammalian Virology, with Dr. André

Bianchi, Prof. Dr. Jan van Oirschot and Prof. Dr. Albert

Osterhaus. June 1997 – September 2001

Influenza A virus. Variability: - Reassortment - Antigenic shift- Antigenic drift

When a single cell is infected by two or more different influenza viruses, reassortment, gene exchange between parental viruses, gives rise to new progeny viruses. 8 segments → 254 combinations

Influenza A virus. - Reassortment - Antigenic shift- Antigenic drift

Phylogenetic tree of influenza HA subtypes

10% of residues

BH8

H12

H9H1

H2

H5

H6

H11

H13

H3

H4

H14H10

H7

H15

H16

16 subtypes of HA

9 subtypes of NA

144 possible combinations HxNy

Seen in humans:

H1, H2 and H3

Others sporadic

N1 and N2

Reservoir of influenza A virusesReservoir of Influenza viruses

1993 The Netherlands,

son of a pig farm

worker with reassortant

H3N2 from pigs

24 April 2009 Mexico,

H1N1 first disease

outbreak notice →

pandemic

Hey,

who

said

that?

He says ice-cream made

him feel better, and Thank God

has now recovered full

health.But the rest

of the planet has a quick –

paced pandemic marching on….

'Patient Zero' in Swine Flu Outbreak Identified as 5-Year-Old Mexican Boy:

Edgar Hernandez

Gene . Origin .

1 PB2 polymerase avian North American

2 PB1 polymerase human H3N2

3 PA polymerase avian North American

4 HA hemagglutinin swine North American

5 NP nucleoprotein swine North American

6 NA neuraminidase swine Eurasian

7 NS nonstructural swine North American

8 M M protein swine Eurasian

2009 H1N1genome:

Reservoir of influenza A virusesReservoir of Influenza viruses40 million death

worldwide, 2.5%

2 million, <0.1% 1 million, <0.1% ¼ - ½ million

death a year

worldwide,

<0.1%

?

H1N1

PB1

H1N1

¼ - ½ million

death a year

worldwide ,

0.03%?

Reasons to study flu and universal vaccines in pigs:

• Pigs are a good flu vaccine model. Pigs can be naturally

infected by avian, swine and human flu viruses

• Pigs are a potential intermediate host and ¨mixing vessel¨

for flu viruses.

• Swine flu causes considerable economic loss to the pig

farming industry

Protection in mice after vaccination

with M2e-HBc protein

HBcHepatitis B

core protein

Injected as

protein

M2e

A universal influenza A vaccine based on the extracellular domain of the M2

protein. Nat. Med. 1999;5(10):1157–1163.

Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers, Walter.

Design of potential universal vaccine

M2e

A universal influenza A vaccine based

on the extracellular domain of the M2

protein. Neirynck S, Deroo T, Saelens

X, Vanlandschoot P, Jou WM, Fiers W.

Nat. Med. 1999;5(10):1157–1163.

Protection in mice after vaccination

with M2e-HBc protein

M2eHBc

Hepatitis B

core protein

Injected as

protein

DNA construct expressing fusion protein of influenza

extracellular part of M2 (M2e,) fused to Nucleoprotein

NPInfluenza

Nucleoprotein

Injected as

DNA plasmid

Conserved target for

antibody mediated immunity

Conserved target for cell

mediated immunity (T-helper

and Cytotoxic T-cells)

Vaccination-challenge experimental design

Day 0, 21, 42:

6 Pigs vaccinated with M2eHBc fusion protein

6 M2eHBc fusion protein + adjuvant

6 M2e-NP DNA

6 non-vaccinated controls PBS

Day 70, 4 weeks later, challenge with 108 TCID50 A/Swine/Best/96 (H1N1)

Clinical signs

Laboured breathingAbdominal breathingAnorexiaApathyCoughing

M2eHBc fusion protein (▲,a)

M2eHBc fusion protein + adjuvant (,b)

M2eNP DNA (○,c)3 pigs died day 1/2

control pigs (□)

Antibody titers

M2eHBc fusion protein (▲,a)

M2eHBc fusion protein + adjuvant (,b)

M2eNP DNA (○,c)3 pigs died day ½ p.i.

control pigs (□)

Lymphoproliferation Lymphoproliferation(cell-mediated immunity)

M2eHBc fusion protein (▲,a)

M2eHBc fusion protein + adjuvant (,b)

M2eNP DNA (○,c)3 pigs died day ½ p.i.

control pigs (□)

T-lymphocyte populationsin the lung

M2eHBc fusion protein (▲,a)

M2eHBc fusion protein + adjuvant (,b)

M2eNP DNA (○,c)2 pigs died day ½ p.i.

control pigs (□)

Significantly more T-helpercells (and cytototoxic T-cells) after challenge, in lung fluid of DNA vaccinated pigs.

• extensive influenza virus

infection

• non-neutralising M2e

antibodies

• the NP-specific T-helper

cells

• all act together to stimulate

macrophages in the lung,

leading to an over-

induction of cytokines

(TNF-), inflammation and

severe clinical signs.

Hypothesis to explain severe clinical signs after challenge infection:

FcR

MHC II

APC

MHC II

APC

FcR

Th cell

NP! NP

M2e Ab

Also, in 1918 those 5 to 14 years of age

accounted for a disproportionate number of

influenza cases, but had a much lower death

rate from influenza and pneumonia than other

age groups. To explain this pattern, we must

look beyond properties of the virus to host and

environmental factors, possibly including

immunopathology (e.g., antibody-dependent

infection enhancement associated with prior

virus exposures (as in dengue)) and exposure

to risk cofactors such as coinfecting agents,

medications, and environmental agents.

1918 Influenza: the Mother of All Pandemics

Jeffery K. Taubenberger* and David M. Morens†

*Armed Forces Institute of Pathology, Rockville, Maryland, USA; and †National Institutes of

Health, Bethesda, Maryland, USA

Emerging Inf. Dis. Jan 2006

40 million death worldwide

Death rate per 100,000 persons

Conclusion

• Non-neutralizing antibodies to M2e

seem to cause exacerbation of disease

during influenza infection

• Especially in combination with cellular

immunity

• We should be very careful before

using M2e in people.

• Unfortunately this could not be

investigated further

M2e flu vaccine trial Acambis , January 2008Phase I clinical trial: 79 volunteers.Response measured in 90% of volunteers, and no adverse effects seen.

Adverse effects upon infection???Phase II clinical trial

M2e vaccine criticism, Company does not really expect a usable vaccine but does research on universal vaccines only to attract attention and raise the value of stocks.

WHO vaccine research, September 2009, results presented cast doubt on the feasibility of using safely M2e as an immunogen in humans.

Interest in M2e and universal vaccines has increased,

especially since the 2009 H1N1 outbreak.

Perhaps we will hear more about this vaccine in the future.

Or perhaps not….

Universal flu vaccine test in pigs

Dr. Paul Heinen

Facultad de Ciencias Exactas y Naturales

Universidad Nacional de Asunción

paulheinen@yahoo.com.ar